Host-directed therapies for salmonella and francisella infection

Abstract

The invention relates to small molecule compounds that are useful as antimicrobial agents, pharmaceutical compositions comprising the compounds, and the use of the compounds in the treatment of subjects with microbial infections.

Description

HOST-DIRECTED THERAPIES FOR SALMONELLA AND FRANCISELLA INFECTION FIELD OF THE INVENTION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 684,178 filed on August 16, 2024, the entire contents of which are hereby incorporated by reference.

[0002] The invention relates to small molecule compounds that are useful as antimicrobial agents. The invention also relates to the use of pharmaceutical compositions comprising these compounds in the treatment of patients with bacterial infections. STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY- SPONSORED RESEARCH

[0003] The invention was made with government support under Grant Numbers AI125147 and AI123692, awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0004] Antimicrobial resistance is a major healthcare problem worldwide. The World Health Organization explains that the emergence and spread of drug-resistant pathogens threatens the world’s ability to treat common infections and to perform life-saving procedures (e.g., chemotherapy, caesarean sections, organ transplantation). Drug-resistant pathogens also threaten our food source with food-borne contamination and could be applied as the highest-level bioterrorism agents, according to the Centers for Disease Control. Two such problematic pathogens are gram-negative bacteria Salmonella enterica and Francisella tularensis, which can cause major illnesses in humans and other animals and have shown antimicrobial resistance.

[0005] Thus, there is a critical need for the development of novel antimicrobial compounds. Salmonella enterica

[0006] Salmonellosis is a major bacterial enteric illness caused by food and water that is contaminated with gram-negative bacteria Salmonella enterica. This illness can result in death and irreparable gastrointestinal damage.

[0007] Although there are known antimicrobial treatments for salmonellosis, decades-long overuse or misuse of antimicrobials has resulted in antimicrobial resistance in typhoidal and non-typhoidal serotypes of Salmonella.

[0008] Studies of typhoidal serotypes of Salmonella show resistance against various antimicrobials. For example, clinical isolates of S. Typhi show large-scale resistance against first-line antimicrobials (chloramphenicol, ampicillin, and trimethoprim- sulfamethoxazole). Further, reports of isolates of S. Typhi in Asia show resistance to first, second, and third-line antimicrobials, including cephalosporins. Some S. Typhi strains are multi-drug resistant (MDR).

[0009] Drug resistance is increasingly reported also in non-typhoidal serotypes of Salmonella. Approximately ~10% of reported non-typhoidal cases show resistance to ciprofloxacin, a commonly available second-line fluoroquinolone used to treat enteric fever.

[0010] Not only is there increasing antimicrobial resistance to commonly available therapies, but there is also a dearth of novel antimicrobials against gram-negative pathogens. This scarcity is the result of a slowing antimicrobial pipeline over the last decade as well as toxicity concerns of existing last-line therapies. Thus, new treatment options that are effective against resistant strains of S. enterica are needed. Francisella tularensis

[0011] Tularemia, also known as rabbit fever, is an illness caused by gram-negative, facultative bacteria Francisella tularensis. F. tularensis is a zoonotic pathogen which mainly affects rodents and lagomorphs but also infects birds, domestic animals, and humans.

[0012] There are three subspecies of F. tularensis, in increasing order of concern to human health: F. tularensis subsp. mediasiatica, F. tularensis subsp. holarctica, and F. tularensis subsp. tularensis. The first, F. tularensis subsp. mediasiatica, rarely causes disease in humans. The second, F. tularensis subsp. holarctica, has an infectious dose of fewer than 1,000 bacteria, and infections are rarely fatal. The third, F. tularensis subsp. tularensis is the most virulent of the subspecies, with an infectious dose of fewer than 10 bacteria and a mortality rate approaching 60%. Because of this low infectious dose, combined with its ability to be easily aerosolized, the CDC recognizes F. tularensis as a Tier 1 Select Agent with potential to be weaponized.

[0013] Transmission of F. tularensis to humans can occur through multiple routes, including arthropod vectors (e.g., ticks), direct contact with infected animal tissue, ingestion of contaminated food or water, and inhalation of aerosolized organisms.

[0014] Tularemia is difficult to diagnose for various reasons. First, clinical symptoms manifest differently depending on the route of transmission. Second, symptoms can be non-specific (e.g., fever and a severe cough for pneumonic tularemia).

[0015] Tularemia infections have been difficult to treat due to the intracellular niche of F. tularensis as well as its intrinsic resistance to many antimicrobials, such as β-lactams, macrolides, linezolid, and clindamycin. As a result, prophylaxis and treatment of tularemia rely on a limited number of effective antimicrobials, and these are associated with significant adverse effects. First-line treatments against tularemia include aminoglycosides, such as gentamicin or streptomycin. These drugs are occasionally associated with severe toxicity and can only be administered parenterally. Gentamicin causes minimal toxicity when administered orally but can cause ototoxicity and nephrotoxicity when administered intravenously or intramuscularly, especially with longer treatments. Further, acute toxicity leading to death has been shown with concentrations as low as 30 mg / kg in monkeys. Alternative tularemia treatments include ciprofloxacin, imipenem-cilastatin, doxycycline, and chloramphenicol. These treatments also have significant adverse reactions such as tendinitis, tendon rupture, arthralgia, myalgia, peripheral neuropathy, and central nervous system effects.

[0016] In addition to the significant adverse effects caused by known treatments of tularemia, post-antimicrobial relapses have been reported. Indeed, post-antimicrobial relapses of up to 33% have been reported with fluoroquinolones, tetracyclines, and chloramphenicol treatments. These post-antimicrobial relapses pose a risk of drug resistance. Selection for resistant F. tularensis strains has been shown in vitro with constant increasing concentrations of ciprofloxacin. For these reasons—toxicity associated with first-line treatments, treatment failures and relapses, and potential emergence of antimicrobial resistance—new tularemia treatment approaches are needed. Host-Directed Therapeutics

[0017] Alternatives to known treatments for bacterial infections are host directed therapies (HDTs, also known as host-targeted therapies). HDTs are advantageous because theysuppress pathogenic bacteria indirectly by targeting host-encoded functions necessary for bacterial infection, replication, virulence, and pathogenesis. For example, HDTs can work by interfering with a host mechanism the bacteria require for growth or by stimulating host immune mechanisms that bacteria evade (e.g., autophagy).

[0018] Because HDTs target host cells (e.g., macrophages and intestinal epithelial cells) instead of bacteria, HDTs counter the problem of antimicrobial resistance by relieving the direct selective pressure on bacteria that typically drives multi drug resistance.

[0019] Several drugs are being evaluated for HDTs against bacterial infections. For example, metformin, which is widely used to treat type 2 diabetes, has been shown to control Mycobacterium tuberculosis infections. M. tuberculosis works by reprogramming host cell metabolism and inhibiting autophagy; this protects M. tuberculosis from autophagic killing. Metformin treatment interrupts this infectious mechanism by inhibiting the mitochondrial respiratory chain, activation of 5ʹ-adenosine monophosphate-activated protein kinase (AMPK), and subsequent induction of autophagy and promotion of phagosome maturation. This ultimately results in M. tuberculosis being captured within an autophagolysosome and killed by reactive oxygen species and autophagolysosomal enzymes.

[0020] Another drug that is being evaluated for HDTs is AR-12 (originally named OSU- 03012):Although AR-12 was initially developed as a cancer chemotherapeutic agent and reached clinical trials and FDA IND approval for that application, studies have shown that it also has broad spectrum host-directed activity against a range of pathogens including bacteria, parasites, viruses, and fungi. For example, studies have shown that AR-12 controls bacteria (e.g., S. enterica, F. tularensis) and protists (e.g., Leishmania donovani) with the use of acetalated-dextran microparticles (Ace-DEX MPs), which can passively target phagocyticcells like macrophages. Studies have also indicated that AR-12 suppresses expression of ER chaperone protein GRP78 to inhibit ampicillin- and kanamycin-resistant E. coli, multi- drug resistant (MDR) Neisseria gonorrhoeae, and SARS-CoV-2. Other studies have shown that AR-12 promotes autophagy in macrophages. For example, AR-12 has been shown to clear intracellular F. novicida via autophagy induction.

[0021] Studies have shown that AR-12 induces autophagy via the Akt pathway and by inhibiting chaperone proteins, such as GRP 78. This is pertinent to S. enterica infections, because S. enterica bacteria use several strategies to manipulate host cell machinery and evade immune cell detection and autophagy. Other studies have shown that AR-12 promotes autophagy in macrophages. For example, AR-12 cleared intracellular F. novicida via autophagy induction, as autophagy inhibition with 3-methyladinine (3-MA) rescued F. novicida intracellular growth in AR-12-treated THP-1 cells.

[0022] The present invention provides chemical compounds that are analogs of AR-12, but which have reduced host cell toxicity and enhanced bacterial clearance against S. enterica and F. tularensis compared to AR-12. BRIEF SUMMARY OF THE INVENTION

[0023] One aspect of the invention is directed to novel chemical compounds of Formula (I):wherein: A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6; R1is -H, -F, -Cl, -Br, or -I;R2is -NO2, -NHR7,R3 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R4is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl; or a substituted or unsubstituted aryl; R5is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl; or a substituted or unsubstituted aryl; R6 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R7 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or; R8 is -H, -F, -Cl, -Br, -I, -a substituted or unsubstituted C1-C6 alkyl, -OR12, or -NR13N14; R9is -H, -F, -Cl, -Br, -I, -a substituted or unsubstituted C1-C6alkyl, -OR12, or -NR13N14; R10is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or phenyl; R11 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; R12 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or phenyl; R13is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, acarbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

[0024] Another aspect of the invention is directed to novel pharmaceutical compositions comprising compounds of Formula (I).

[0025] Another aspect of the invention is methods of treating infections caused by bacterial pathogens, comprising administration of a pharmaceutical composition comprising compounds of Formula (I). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG.1. Process flow of screening methodology and hit selection for evaluation of AR-12 analogs against salmonella infections.

[0027] FIG.2. Autophagic flux as measured by fluorescence intensity of green autophagy dye in salmonella-infected RAW264.7 macrophages measured via flow cytometry.

[0028] FIG.3. Diagram of three-round screening process of AR-12 analog compounds evaluated for host-directed antibacterial activity against intracellular F. tularensis.

[0029] FIG.4. Results from initial 10µM screen to determine AR-12 analog compound effect on intracellular LVS bacterial burden at 24h normalized to DMSO control.

[0030] FIG.5. Direct effect of AR-12 analog compounds which advanced from primary screen on LVS growth in defined medium at 24h normalized to DMSO control.

[0031] FIG.6. Structures of twelve AR-12 analog compounds identified in screen for host- directed antibacterial activity against intracellular F. tularensis.DETAILED DESCRIPTION OF THE INVENTION

[0032] One aspect of the invention is directed to novel chemical compounds of Formula (I):wherein: A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6; R1is -H, -F, -Cl, -Br, or -I; R2is -NO2, -NHR7,R3 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R4is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl; or a substituted or unsubstituted aryl; R5is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl; or a substituted or unsubstituted aryl; R6 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl;R7is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or; R8 is -H, -F, -Cl, -Br, -I, -a substituted or unsubstituted C1-C6 alkyl, -OR12, or -NR13N14; R9 is -H, -F, -Cl, -Br, -I, -a substituted or unsubstituted C1-C6 alkyl, -OR12, or -NR13N14; R10 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or phenyl; R11is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; R12 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or phenyl; R13 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

[0033] The novel chemical compounds of Formula (I) are analogs of AR-12. These compounds of Formula (I) have improved activity as compared to AR-12, because they show reduced host cell toxicity and enhanced bacterial clearance against pathogens, such as S. enterica and F. tularensis. Without wishing to be bound by theory, it is believed thatthe antimicrobial activity of the compounds of Formula (I) is directed against intracellular bacteria and not directly against the bacteria, which can mitigate development of drug resistance.

[0034] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6; R1is -H, -F, -Cl, -Br, or -I; R2 is -NO2, -NHR7,R3 is -H, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCl3, -CHCl2, -CH2Cl, -CBr3, -CHBr2, - CH2Br, -CI3, -CHI2, -CH2I, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R4is -H, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCl3, -CHCl2, -CH2Cl, -CBr3, -CHBr2, - CH2Br, -CI3, -CHI2, -CH2I, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R5 is -H, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCl3, -CHCl2, -CH2Cl, -CBr3, -CHBr2, - CH2Br, -CI3, -CHI2, -CH2I, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R6 is -H, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCl3, -CHCl2, -CH2Cl, -CBr3, -CHBr2, - CH2Br, -CI3, -CHI2, -CH2I, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl;R7is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, , or; R8 is -H, -F, -Cl, -Br, -I, -CF3, -CCl3, -CBr3, -CI3, methyl, ethyl, n-propyl, isopropyl, n- butyl, sec-butyl, tert-butyl, -OR12, or -NR13N14; R9is -H, -F, -Cl, -Br, -I, -CF3, -CCl3, -CBr3, -CI3, methyl, ethyl, n-propyl, isopropyl, n- butyl, sec-butyl, tert-butyl, -OR12, or -NR13N14; R10is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or phenyl; R11is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; R12 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or phenyl; R13 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

[0035] In one embodiment, the present invention relates to compounds of Formula (I), wherein:A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6; R1is -H, -F, -Cl, -Br, or -I; R2 is -NO2, -NHR7,R3 is -H, -F, -Cl, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted naphthyl, a substituted or unsubstituted anthryl, a substituted or unsubstituted phenanthryl, or a substituted or unsubstituted pyrenyl; R4 is -H, -F, -Cl, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted naphthyl, a substituted or unsubstituted anthryl, a substituted or unsubstituted phenanthryl, or a substituted or unsubstituted pyrenyl; R5is -H, -F, -Cl, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted naphthyl, a substituted or unsubstituted anthryl, a substituted or unsubstituted phenanthryl, or a substituted or unsubstituted pyrenyl; R6is -H, -F, -Cl, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted naphthyl, a substituted or unsubstituted anthryl, a substituted or unsubstituted phenanthryl, or a substituted or unsubstituted pyrenyl; R7is -H, methyl, ethyl,, R8 is -H, -F, -Cl, -CF3, methyl, ethyl, -OR12, or -NR13N14;R9is -H, -F, -Cl, -CF3, methyl, ethyl, -OR12, or -NR13N14; R10 is -H, methyl, ethyl, or phenyl; R11 is -H, methyl, ethyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert- butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9- fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p- toluenesulfonyl (tosyl, Ts) group; R12is -H, methyl, ethyl, or phenyl; R13is -H, methyl, ethyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert- butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9- fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p- toluenesulfonyl (tosyl, Ts) group; and R14 is -H, methyl, ethyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert- butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9- fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p- toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

[0036] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6; R1is -H or -F;R2is -NO2, -NHR7,R3 is -H, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl; R4is -H, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl; R5is --H, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl; R6 is -H, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl; R7 is -H,R8is -H or -NR13N14; R9is -H or -NR13N14; R10 is -H, methyl, or phenyl; R11 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; R13 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl(BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

[0037] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6; R1is -H or -F; R2is -NO2, -NHR7,R3 is -H, -CF3, methyl, ethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl; R4is -H, -CF3, methyl, ethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl; R5is -H, -CF3, methyl, ethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl; R6 is -H, -CF3, methyl, ethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl; R7 is -H,R8 is -H or -NR13N14; R9is -H or -NR13N14;R10is -H, methyl, or phenyl; R11 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; R13 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

[0038] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6; R1 is -H or -F; R2is -NO2, -NHR7,R3is -H, -CF3, methyl, ethyl, cyclopropyl, or a substituted or unsubstituted phenyl; R4 is -H, -CF3, or cyclohexyl;R5is -H, -CF3, or cyclohexyl; R6 is -H, -CF3, methyl, ethyl, cyclopropyl, or a substituted or unsubstituted phenyl; R7 is -H,R8is -H or -NR13N14; R9 is -H or -NR13N14; R10 is -H, methyl, or phenyl; R11 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; R13 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

[0039] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is CR3; B is CR4; D is N;E is CR6; R1 is -H or -F; R2 is -NO2, -NHR7, orR3is -H or -CF3; R4is -H or -CF3; R6 is -H, -CF3, phenyl, or phenyl that is substituted with one or more substituents selected from the group consisting of -F, -Cl, -Br, -I, -CF3, phenyl, 2-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl, or 4-(trifluoromethyl)phenyl; R7 is -H orR8is -H or -NR13N14; R10 is -H, methyl, or phenyl; R13 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

[0040] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is CR3;B is CR4; D is CR5; E is N; R1is -H or -F; R2is -NO2or -NHR7; R3 is -H or -CF3; R4 is -H or -CF3; R5is -H, -CF3, or cyclohexyl; R7is -H orand R10 is -H, methyl, or phenyl; or a pharmaceutically acceptable salt thereof.

[0041] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N; B is N; D is CR5; E is CR6; R1 is -H or -F; R2is -NO2, -NHR7, orR5 is -H or -CF3;R6is -H, -CF3, phenyl, or phenyl that is substituted with one or more substituents selected from the group consisting of -F, -Cl, -Br, -I, -CF3, phenyl, 2-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl, or 4-(trifluoromethyl)phenyl;R7 is -H orR9is -H or -NR13N14; R10is -H, methyl, or phenyl; R13 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

[0042] In one embodiment, the present invention relates to compounds of Formula (I), wherein: A is N; B is CR4; D is CR5; E is CR6; R1is -H or -F; R2is -NO2or -NHR7; R4 is -H or -CF3;R5is -H or -CF3; R6 is -H, -CF3, methyl, ethyl, or cyclopropyl; RR11is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

[0043] One embodiment of the invention is directed to compounds of Formula (I) selected from the group consisting of: ;or a pharmaceutically acceptable salt thereof.

[0044] Another aspect of the invention is directed to novel pharmaceutical compositions comprising a therapeutically effective amount of at least one compound of Formula (I) and at least one pharmaceutically acceptable excipient. Examples of suitable pharmaceutically acceptable excipients are antioxidants; preservatives (e.g., ethylenediaminetetraacetic acid, “EDTA”; ethyleneglycol-bis(β-aminoethyl)-N,N,N’,N’-tetraacetic acid, “EGTA”; butylated hydroxyanisole, “BHA”; and butylated hydroxytoluene, “BHT”); coloring agents; flavoring agents; emulsifying agents; suspending agents; solvents; fillers; bulking agents; buffers (e.g., phosphates, carbonates, and citrates); delivery vehicles; binders; disintegrants; diluents; glidants; lubricants; pharmaceutical adjuvants; and the like, which are known to a person skilled in the art.

[0045] The pharmaceutical compositions of the invention may be prepared by any of the well- known techniques of pharmacy, such as effective formulation and administration procedures.

[0046] The compounds of Formula (I) exhibit resensitization of drug resistance with co- delivery with conventional antimicrobials. The compounds of Formula (I) also exhibit mathematical synergy with conventional antimicrobial therapies, permitting a reduced concentration of the conventional antimicrobial therapies. Accordingly, the invention also includes pharmaceutical compositions comprising (a) a therapeutically effective amount of a compound of Formula (I); (b) a therapeutically effective amount of at least one other pharmaceutically active agent selected from the group consisting of antibacterial compounds, antiviral compounds, antifungal compounds, antiparasitic compounds, and combinations thereof; and (c) a pharmaceutically acceptable excipient. Methods for testing resensitization of drug resistance by administering AR-12 with co-delivery of conventional antimicrobials (e.g., streptomycin, ampicillin) can be found in M.S.H. Zahid et al.,“Overcoming reduced antibiotic susceptibility in intracellular Salmonella enterica serovar Typhimurium using AR-12,” FEMS Microbiology Letters, 368, 2021, 1-9. The entire contents of this disclosure are incorporated by reference herein.

[0047] In one embodiment of the invention, the at least one other pharmaceutically active agent for use in the pharmaceutical compositions of the invention may be selected from the group consisting of beta-lactams, floroquinolones, macrolides, aminoglycosides, tetracyclines, and mixtures thereof.

[0048] In one embodiment of the invention, the at least one other pharmaceutically active agent for use in the pharmaceutical compositions of the invention may be selected from the group consisting of gentamicin, streptomycin, rapamycin, kanamycin, hygromycin, clindamycin, penicillin, ampicillin, chloroquine, chloramphenicol, linezolid, ciprofloxacin, imipenem, cilastatin, doxycycline, and mixtures thereof.

[0049] Yet another aspect of the invention is a new method of treating infections caused by a microbial species, comprising administration of a therapeutically effective dose of a compound of Formula (I).

[0050] The compounds of Formula (I) may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The compounds of Formula (I) and pharmaceutical compositions comprising a therapeutically effective amount of at least one compound of Formula (I) may be administered orally, rectally, vaginally, parenterally, intramuscularly, intravenously, subcutaneously, transdermally, topically, or by inhalation.

[0051] The dosage regimen for the compounds of the invention or the pharmaceutical compositions of the invention is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed.

[0052] In many instances, the administration of the compounds of the invention or the pharmaceutical compositions of the invention will be repeated a plurality of times in a day. Multiple doses per day typically may be used to increase the total daily dose, if desired.

[0053] The compounds of Formula (I) can be used, alone or in combination with other pharmaceutically active agents, in the treatment of an infection caused by a microbial species. At least one compound of Formula (I) and at least one other pharmaceutically active agent may be may be administered simultaneously (either in the same dosage form or in separate dosage forms) or sequentially.

[0054] The phrases “concurrent administration,” “co-administration,” “simultaneous administration,” and “administered simultaneously” mean that the compounds are administered in combination.

[0055] In one embodiment of the invention, a subject in need of treatment of an infection caused by a microbial species with a compound of Formula (I) is an animal. Examples of animals that may be treated according to the invention include fish, amphibians, and mammals. Examples of mammals that may be treated according to the invention include humans, primates, horses, sheep, pigs, cows, mice, rats, rabbits, dogs, and cats.

[0056] In one embodiment of the invention, the new method of treating an infection caused by a microbial species comprises administering to a subject in need thereof a therapeutically effective amount of at least one compound of Formula (I).

[0057] In one embodiment of the invention, the new method of treating an infection caused by a microbial species comprises administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of Formula (I).

[0058] In one embodiment of the invention, the new method of treating an infection caused by a microbial species comprises administering to a subject in need thereof a therapeutically effective amount of at least one compound of Formula (I) and a therapeutically effective amount of at least one other pharmaceutically active compound selected from the group consisting of an antibacterial compound, an antiviral compound, an antifungal compound, an antiparasitic compound, and combinations thereof.

[0059] In one embodiment of the invention, the new method of treating an infection caused by a microbial species comprises administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of Formula (I) and therapeutically effective amount of at least one otherpharmaceutically active compound selected from the group consisting of an antibacterial compound, an antiviral compound, an antifungal compound, an antiparasitic compound, and combinations thereof.

[0060] In one embodiment of the invention, the new method of treating an infection caused by a microbial species comprises co-administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of Formula (I) and a pharmaceutical composition comprising a therapeutically effective amount of at least one other pharmaceutically active compound selected from the group consisting of an antibacterial compounds, an antiviral compound, an antifungal compound, an antiparasitic compound, and combinations thereof.

[0061] In one embodiment of the invention, the at least one other pharmaceutically active agent for use in the methods of the invention may be selected from the group consisting of beta- lactams, floroquinolones, macrolides, aminoglycosides, tetracyclines, and mixtures thereof.

[0062] In one embodiment of the invention, the at least one other pharmaceutically active agent for use in the methods of the invention may be selected from the group consisting of gentamicin, streptomycin, rapamycin, kanamycin, hygromycin, clindamycin, penicillin, ampicillin, chloroquine, chloramphenicol, linezolid, ciprofloxacin, imipenem, cilastatin, doxycycline, and mixtures thereof.

[0063] In one embodiment of the invention, the infection is caused by Salmonella enterica.

[0064] In one embodiment of the invention, the infection is caused by a Francisella tularensis. Definitions

[0065] The term “alkyl” refers to straight or branched chain alkyl radicals having 1 to 20 carbon atoms, and “substituted alkyl” refers to alkyl radicals further bearing one or more substituents.

[0066] The term “C1-C6alkyl” refers to straight or branched chain alkyl radicals having 1 to 6 carbon atoms, and “substituted C1-C6 alkyl” refers to C1-C6 alkyl radicals further bearing one or more substituents.

[0067] The term “cycloalkyl” refers to cyclic ring-containing hydrocarbon moieties containing 3 to 20 carbon atoms, and “substituted cycloalkyl” refers to cycloalkyl moieties further bearing one or more substituents.

[0068] The term “C3-C10 cycloalkyl” refers to cyclic ring-containing hydrocarbon moieties containing 3 to 10 carbon atoms, and “substituted C3-C10cycloalkyl” refers to C3-C10cycloalkyl moieties further bearing one or more substituents.

[0069] As used herein, the term “aryl” refers to aromatic groups having 6 to 24 carbon atoms, and “substituted aryl” refers to aryl groups further bearing one or more substituents. Examples of aryl groups are phenyl, naphthyl, fluorenyl, anthryl, phenanthryl, and pyrenyl.

[0070] The term “pharmaceutically acceptable” refers to a non-toxic material that does not interfere with the effectiveness of the active ingredient(s).

[0071] The term “therapeutically effective amount” means an amount of a compound of the invention that (i) treats or prevents a particular disease, condition, or disorder described herein, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder described herein, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

[0072] Substituted alkyl groups are substituted with one or more substituents selected from the group consisting of -F, -Cl, -Br, -I, -CN, -NO2, -ORa, -SRb, -NRcRd, phenyl, pyridyl, -CHO, -COORe, -CO(NRfRg); wherein each of Ra, Rb, Rc, Rd, Re, Rf, and Rgare independently selected from H or C1-C6 alkyl.

[0073] Substituted cycloalkyl groups and substituted aryl groups are substituted with one or more substituents selected from the group consisting of C1-C6 alkyl, -F, -Cl, -Br, -I, -CN, - NO2, -ORa, -SRb, -NRcRd, phenyl, o-toluyl, m-toluyl, p-toluyl, 2-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl, 4-(trifluoromethyl)phenyl, pyridyl, -CF3, -CCl3, -CBr3, -CI3, - CHO, -COORe, -CO(NRfRg); wherein each of Ra, Rb, Rc, Rd, Re, Rf, and Rg are independently selected from H or C1-C6alkyl.

[0074] The term “salt” refers to acid addition salts, including Cl-, Br-, I-, NO2 -, HSO4 -,SO4-, HPO4-, PO42-, ethanesulfonate, trifluromethane sulfate, p-toluenesulfonate,benzenesulfonate, salicylate, propionate, ascorbate, aspartate, fumarate, galactarate, maleate, citrate, glutamate, glycolate, lactate, malate, maleate, tartrate, oxalate, succinate, and the like.

[0075] Where a salt is intended to be administered to a patient (as opposed to, for example, being used in an in vitro context), the salt preferably is pharmaceutically acceptable. The term “pharmaceutically acceptable salt” refers to a salt prepared by combining a compound of Formula (I) with an acid whose anion, or a base whose cation, is generally considered suitable for human consumption. For use in medicine, the salts of the compounds of this invention are non-toxic “pharmaceutically acceptable salts.” Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid.

[0076] As used herein, the term “Formula (I)” may be referred to as a “compound(s) of the invention,” “the invention,” and “compound of Formula I.” Such terms are also defined to include all forms of the compound of Formula (I), including hydrates, solvates, isomers, crystalline and non-crystalline forms, isomorphs, polymorphs, and metabolites thereof. For example, the compounds of the invention, or pharmaceutically acceptable salts thereof, may exist in unsolvated and solvated forms. When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water / solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm. Examples

[0077] In the development of the compounds of Formula (I), various AR-12 analogs were evaluated for their activity against S. enterica and F. tularensis. The evaluation of the AR- 12 analogs considered activity (MIC) and selectivity (which takes into account host cell cytotoxicity). Further, using a mouse model, a selection of the AR-12 analogs was evaluated via oral infection and delivery. Resensitization of resistant strains and reduction in the emergence of drug resistance was shown in vitro.Example 1 – Evaluation of AR-12 Analogs for S. Enterica

[0078] The process flow of screening methodology and hit selection of AR-12 analog compounds for S. enterica is summarized in FIG.1.

[0079] Bacterial Strains and Mammalian Cell Lines. Susceptible (ATCC 700720) and MDR (ATCC 700408) strains of S. enterica serovar Typhimurium were obtained from American Type Culture Collection (ATCC Manassas, VA) and cultured in nutrient broth and Luria-Bertani (LB) broth (BD Difco, Detroit, MA), respectively. Murine macrophage cell line RAW264.7 (ATCC TIB-71) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (GIBCO) and 1% penicillin-streptomycin (P / S) (GIBCO). Cells were cultured at 37 °C and 5% CO2and used up to passage 10 once received from ATCC.

[0080] Initial Screening Using Fluorescence Assay. Preliminary screening of 67 compounds was performed using a fluorescence-based high throughput assay. RAW264.7 cells were seeded in a black well 96-well plate and were infected with pBR-RFP modified S. Typhimurium at multiplicity of infection (MOI) of 50 over 1 hr at 37 °C. Infected cells were treated with gentamicin at 100 µg / ml for 1 hr at 37 °C. Cells were washed with antibiotic-free medium and treated with compounds for 24 hr. Post-treatment, cells were washed and stained with CellTracker Green (ThermoScientific), fixed with 4% formaldehyde for 20 min at 37 °C, and stained with 0.1 ug / ml DAPI for 20 min. Fluorescence intensity of CellTracker (536 nm), 4’,6-diamidino-2-phenylindole (DAPI) (447 nm), and red fluorescent protein (RFP) (624 nm) were acquired on ImageXpress Micro 4 High-Content System (Molecular Devices), and bacterial viability was quantified using MetaXpress image analysis software (Molecular Devices).

[0081] Intracellular CFU Assay. RAW264.7 cells were seeded at 3x104cells / well in a 96- well plate with and maintained at 37 °C for 20 hours. A single colony of susceptible or MDR S. Typhimurium was inoculated and cultured overnight in nutrient broth and diluted to obtain a log-phase culture stock with an adjusted optical density of 0.6, equivalent to a concentration of 3x108CFU / mL. The bacterial stock solution was resuspended in antibiotic-free DMEM supplemented with 2% FBS and further diluted to obtain a bacterial concentration of 3x106CFU / mL. Cell media was aspirated and replaced with 100 µL of bacteria culture in DMEM resulting in a MOI of 10. The cells underwent infection for twohours or 30 minutes at 37 °C with susceptible or MDR bacteria, respectively. Media was aspirated from the wells and replaced with DMEM (2% FBS) containing 100 µg / ml gentamicin for one hour at 37 °C to eliminate extracellular bacteria. Following this step, cells were washed once with media and treated for 22 hours with AR-12 analogs at varying concentrations (0.1 - 5 µM) in DMEM (2% FBS, no P / S) containing 10 µg / ml gentamicin. AR-12-treated, uninfected, and dimethylsulfoxide-vehicle (DMSO-vehicle) treated cells were included as controls. Post-treatment, cells were washed with phosphate buffered saline (PBS) and lysed with 0.1% Triton-X100. The lysates containing intracellular bacteria were serially diluted and loaded onto nutrient or LB agar plates (for susceptible and MDR infected cells, respectively). Agar plates were incubated overnight at 37 °C before colony forming units (CFU) from each dilution were counted. Fifty percent inhibitory concentration (IC50) was calculated as the drug concentration that resulted in 50% reduction of intracellular bacteria obtained by curve fitting the normalized CFU against increasing drug concentration.

[0082] Cell Proliferation and Viability Assay. Cell proliferation and viability in the presence of the AR-12 analogs was tested in the macrophage cell line RAW264.7 using a 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells were seeded at 3x104cells / well in 96 well plates in DMEM (10% FBS, 1% P / S). After an overnight incubation at 37 °C, cells were treated with AR-12 analogs at concentrations ranging between 1-100 µM at 37°C for 24 hours. DMSO vehicle was utilized as a control, as all the drugs were reconstituted from DMSO stocks. After 24 hr treatment, media was replaced with 100 µL of 0.6 mg / mL MTT solution, and cells were maintained in culture medium at 37 °C until formazan crystals formed. The MTT solution was then replaced with 100 µL of isopropanol, to dissolve the formazan crystals. Cell viability was determined by measuring absorbance at 560 nm of drug treated cells normalized to untreated DMSO vehicle controls. The absorbance at 670 nm was used as a background correction. Cell viability was plotted against increasing drug concentration to obtain the 50% lethal concentration (LC50), defined as the drug concentration resulting in 50% cell viability.

[0083] Direct Effect on Bacteria. A single colony of susceptible or MDR bacteria was inoculated in nutrient broth and incubated at 37 °C in an orbital shaker set at 200 rpm. The culture was diluted to obtain log-phase bacterial growth after an additional 3 hr incubationat 37 °C and further adjusted to obtain a bacterial stock solution with an absorbance value of 0.6 at 600 nm, equivalent to a concentration of 3x108CFU / mL. The bacterial stock was diluted with nutrient broth to obtain 3x104CFU / well in a 96 well plate. Next, 10 microliters of each AR-12 analog were added to yield a total volume of 100 µL / well with a final concentration of 20 µM. Drug-free and bacteria-free wells were used as controls. The plate was incubated at 37 °C for 22 hours. Percent bacterial viability was determined using optical density measurements at 600 nm, subtracting out bacteria-free absorbance (background) and normalizing to drug-free controls.

[0084] Detection of Autophagy Induction by Flow Cytometry. AR-12 analogs were evaluated for their role in autophagy induction or inhibition using a fluorescence-based flow cytometry assay. RAW264.7 cells were seeded in a 10 cm petri dish at a density of 9.5x105cells in 10 mL of DMEM media (10% FBS, 1% P / S). After an overnight incubation at 37 °C, 5% CO2, cells were infected with susceptible S. Typhimurium (ATCC 700720) at MOI of 10 for 2 hours. Extracellular bacteria were eliminated with a 1-hr treatment of gentamicin at 100 µg / ml. Infected cells were treated with the AR-12 analogs at the IC90concentration for 18 hours. Increased autophagic flux control was produced through treatment with rapamycin (0.05 µM) and chloroquine (10 µM). Decreased autophagic flux control was produced through treatment with PHY34 (10 nM). Following treatment, cells were scraped and filtered through a 70 µm strainer, and stained with the Abcam autophagy detection dye (1:1000 dilution, in phenol red free DMEM) for 30 min at 37 °C. Cells were washed with assay buffer and stained with e450 live / dead stain (1:1000 dilution in PBS) for 20 min at 4 °C. Following a wash with assay buffer, the stained cells were fixed with 0.5% paraformaldehyde (in PBS) for 20 min at 4 °C. The fixed cells were washed and resuspended in assay buffer. All media and buffers were supplemented with 5% FBS while staining and fixing cells for flow cytometry analysis. Lastly, stained cells were measured for viability (405 nm) and autophagy green (488 nm) on NxT Attune (Thermo Fisher Scientific), and data was analyzed using FlowJo (10.7.1).

[0085] Statistical analysis. Data are presented as the mean ± standard deviation. One-way ANOVA followed by t-test to determine statistical significance between groups in the flow cytometry results (p < 0.05 was considered significant).

[0086] Results of Primary Screen. Primary screening of 184 AR-12 analogs was performed using a medium-throughput, fluorescence-based assay with RAW macrophages andsusceptible S. Typhimurium. This screen evaluated the change in intracellular salmonella burden and host cell viability after treatment with AR-12 analog compounds (Table 1). Sixty-seven AR-12 analogs with high potency (Fluor IC50< 1 µM) were selected for secondary screening. Table 1

[0087] Results of Secondary Screen. A secondary screen was performed by assessing intracellular growth of S. Typhimurium in RAW264.7 cells using a CFU assay. The effect of compounds on host cell viability and proliferation was evaluated using MTT assay. Additionally, direct anti-bacterial effect of each compound was assessed by treating planktonic bacteria. There were no significant differences observed in bacterial viability between drug treated and untreated bacteria up to 20 µM of drug, which was the maximum concentration evaluated. The IC50 of the compounds against susceptible S. Typhimurium in macrophage cells ranged between 0.03 to >1 µM and selectivity was observed to range from <7 to >1,124 (Table 2). This secondary screening yielded fourteen AR-12 analog compounds with a selectivity >250. These were more than ten times more selective than parental compound AR-12. These fourteen AR-12 analog compounds were selected for tertiary screening. Table 2

[0088] Results of Tertiary Screen. A tertiary screen was performed by assessing intracellular growth of multi-drug resistant (MDR) S. Typhimurium in RAW264.7 macrophages by CFU assay. Additionally, the direct effect of AR-12 analogs on growth of MDR S. Typhimurium was investigated. The IC50of the compounds against MDR S. Typhimurium in macrophage cells ranged between 0.06 to 1.7 µM and selectivity was observed to range from 32 to >5000 (Table 3). All but one AR-12 analog compounds showed similar potency between susceptible and MDR strains. Table 3

[0089] Effects of Compounds in Autophagy. The potential involvement of the AR-12 analog compounds in utilizing the autophagy pathway for their host directed action against S. Typhimurium was assessed using flow cytometry of infected RAW macrophages after 18 hr treatment. AR-12 demonstrated an increased autophagic signal upon treatment of infected cells compared to the DMSO vehicle control, and all but one of the AR-12 analog compounds exhibited a significantly reduced autophagy signal (FIG.2).

[0090] FIG. 2 shows autophagic flux as measured by fluorescence intensity of green autophagy dye in salmonella-infected RAW264.7 macrophages measured via flow cytometry. Baseline autophagy is observed in DMSO vehicle control. PHY34 (10 nM) was used as a negative control and showed decreased mean fluorescence intensity. Rapamycin (0.5 µM) and chloroquine (10 µM) were used as a positive control and indicated increased mean fluorescence intensity. AR-12 analog compounds were evaluated at 1µM except for 177 and 219 which were evaluated at 2µM to best match their IC90(the concentration at which each compound reduced intracellular salmonella burden by 90%). The data in FIG. 2 is presented as the average and standard deviation of 3 biological replicates. ** P- value<0.01, *** P-value<0.001 with respect to the DMSO vehicle control.Example 2 - Evaluation of AR-12 Analogs for F. Tularensis

[0091] The process flow of screening methodology and hit selection of AR-12 analog compounds for F. tularensis is summarized in FIG.3.

[0092] Bacterial Strains. Live vaccine strain (LVS) was obtained from the CDC (Atlanta, GA). F. tularensis subspecies tularensis SchuS4, a recent clinical isolate, was obtained from BEI Resources (Manassas, VA). All F. tularensis strains were cultured using chocolate agar supplemented with 1% IsoVitaleX (chocolate agar) or Chamberlain’s defined medium (CDM). Luciferase-expressing LVS (LUX) was used in the initial 10mM screen and to obtain the concentration at which F. tularensis intracellular growth was reduced by 50% (IC50). Round 3 titration experiments and OD600experiments (experiments measuring optical density of a sample at a wavelength of 600 nm) were performed using wild-type (WT) F. tularensis Schu S4 and MDR SchuS4 variant strain which harbors a kanamycin resistance transposon and plasmid containing a hygromycin resistance cassette.

[0093] Macrophages. J774A.1 cells were obtained from ATCC (Manassas, VA) and were used in all intracellular bacterial growth assays. J774A.1s were grown in Dulbecco’s modified Eagle’s medium with 4.5g / L glucose (Corning, Corning, NY) supplemented with GlutaMAX (ThermoFisher Scientific, Waltham, MA), sodium pyruvate (ThermoFisher Scientific), and 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA).

[0094] Luminescence-Based Bacterial Growth Assay. LUX-expressing F. tularensis was grown over 3 days on chocolate agar containing 200 mg / mL hygromycin (Cayman Chemical, Ann Arbor, MI). On day 3, bacteria were suspended in CDM and grown overnight. J774A.1 cells were seeded at approximately 30,000 cells in 200mL medium per well into a white bottom 96-well plate, allowed to culture overnight, and infected at an approximate multiplicity of infection (MOI) of 100 bacteria / cell in 50mL per well. At 2 hours post inoculation (hpi), medium was exchanged with medium containing 10mg / mL gentamicin (ThermoFisher Scientific) and 10mM of AR-12 analog compound initially diluted in dimethyl sulfoxide (DMSO). Infected cells were then incubated overnight at 37 °C. At 24 hpi, an Infinite 200M Pro series plate reader (Tecan, Männedorf, Switzerland) was used to read luminescence, indicating bacterial numbers. Drug effect on intracellular F. tularensis was calculated as a percentage of bacterial growth compared to DMSO vehicle control. For the secondary screen, the concentration required to reduce intracellular F.tularensis by 50% (LumIC50) value was measured from best fit curves plotting the percentage of bacterial growth to drug concentration.

[0095] Colony-Forming Unit Bacterial Growth Assay. Titration experiments were performed using three different concentrations of each of the 12 final AR-12 analog compounds of interest against F. tularensis subsp. tularensis Schu S4 WT and MDR strains to determine individual IC50 values. J774A.1 cells were seeded at approximately 50,000 cells in 500µL per well into a 24-well plate and infected at an approximate MOI of 100 in a volume of 250mL per well. At 2 hpi, medium was exchanged with medium containing gentamicin (10µg / mL) and 1, 5, and 10 mM AR-12 analog compound. At 24 hpi, medium was exchanged with 1mL PBS, and cells were scraped from wells using a sterile wooden stick and transferred to a 1.5mL tube. Suspensions were vortexed for 30 seconds to liberate bacteria from the J774A.1 cells. Dilutions were plated onto chocolate agar. After 3 days, colonies were counted and used to calculate percentage of F. tularensis growth compared to that of the DMSO vehicle control. For the final screen, the concentration required to reduce intracellular F. tularensis by 50% (IC50) value was measured from best fit curves plotting the percentage of bacterial growth to drug concentration.

[0096] Impact of AR-12 Analog Compounds on Host Cell Visibility. Cytotoxicity of the AR-12 analog compounds on J774A.1 cells was evaluated using CellTiter-Blue® Cell Viability Assay (Promega, Madison, WI) which measures cell metabolic activity. J774A.1 cells were seeded in a 96 well plate (20,000 cells / well) and allowed to adhere overnight. Macrophages were then treated with AR-12 analog compounds in media (1-100 µM) for 24 hours. Media was replaced with CellTiter-Blue solution according to manufacturer’s specifications and incubated at 37 °C for 2 hours. The fluorescence was measured (excitation 560nm, emission 590nm). Background fluorescence was subtracted and relative fluorescence units (RFU) values were normalized to appropriate DMSO vehicle controls. The concentration required to reduce host cell viability by 50% (LC50) value was measured from best fit curves plotting the relative RFU values against drug concentration. This process was repeated with an extended concentration range (1-500 mM) for select AR- 12 analog compounds.

[0097] Direct Effect of AR-12 Analog Compounds on F. Tularensis Growth. Overnight cultures of F. tularensis were grown in CDM and diluted to an OD600 of 0.12-0.16. Compounds were added to the appropriate final concentration (250, 100, 50, 25, or 10mM),and 200mL was transferred in triplicate into a clear 96-well plate. Cultures were incubated at 37 °C in an Infinite 200M Pro series plate reader (Tecan) with orbital shaking. OD600 readings were taken at 0 and 24 h. For the final screen, the concentration required to reduce free-living F. tularensis by 50% (MIC50) value was measured from best fit curves plotting the percentage of bacterial growth to drug concentration.

[0098] Determination of Therapeutic Indices and Host-Directed Impact. Luminescence therapeutic indices were determined for the 90 AR-12 analog compounds in the secondary screen by dividing the LC50by the LumIC50. Therapeutic indices were determined for the 12 AR-12 analog compounds in the final screen by dividing the LC50 by the IC50. Host- directed impact was calculated for the 12 AR-12 analog compounds in the final screen by dividing the MIC50 by the IC50.

[0099] Screening of AR-12 Analog Compounds. 430 AR-12 analog compounds were evaluated for their potential as host-directed F. tularensis therapeutics with low impact on host cell viability compared to the impact on intracellular bacteria (i.e., high selectivity). Three rounds of screening in a single-blinded manner (FIG.3) were performed on the AR- 12 analog compound library.

[0100] Results of Primary Screen. Primary screening of 430 AR-12 analog compounds for antibacterial activity against intracellular LUX-producing F. tularensis was conducted at 10mM using luminescence as a measure for bacterial numbers (FIG.4). Bacterial growth with AR-12 analog compound treatment was compared to that of the DMSO vehicle control at 24 hpi. Compounds with a MIC70, the concentration by which intracellular growth was reduced by 70%, of less than 10mM advanced to the secondary screen. Ninety AR-12 analog compounds met this criterion (Table 4).

[0101] FIG. 4 shows the results from initial 10µM screen to determine compound effect on intracellular LVS bacterial burden at 24h normalized to DMSO control. Each data point represents the percent LVS intracellular growth from one replicate. The light gray dotted line represents the 70% growth inhibition threshold set for advancing compounds. Light gray bars indicate compounds which advanced to the secondary screen.Table 4

[0102] Results of Secondary Screen. In the secondary screen, the 90 advanced AR-12 analog compounds were tested for cytotoxicity on J774A.1 macrophages (LC50), impact on intracellular LVS growth via luminescence (LumIC50), and LVS growth in CDM at 10mM. LC50and IC50values were used to determine the therapeutic index of each AR-12 analog compound. OD600 experiments were performed to determine direct impact of the drugs onLVS at 10mM (FIG.5). The threshold criteria for AR-12 analog compound advancement for further screening were a therapeutic index of 25, which is 5-times that of AR-12, and a concentration at which F. tularensis growth in defined medium was inhibited by 30% (MIC30) of greater than 10mM. Twelve AR-12 analog compounds met these criteria (FIG. 5; Table 5). Table 5 shows the CFU IC50, MIC50, therapeutic index, and HDT selectivity of the twelve selected AR-12 analog compounds identified in the secondary screen.

[0103] FIG. 5 shows the direct effect of AR-12 analog compounds which advanced from primary screen on LVS growth in defined medium at 24 h normalized to DMSO control. Each data point represents the % LVS growth from one replicate after 10µM treatment with compound. The light gray dotted line represents the 30% growth inhibition threshold set for the secondary screen. Light gray bars indicate compounds which advanced to the final screen. Table 5

[0104] Results of Tertiary Screen. Therapeutic index and direct impact on bacterial growth were determined for each of the twelve selected AR-12 analog compounds against the highly pathogenic F. tularensis subsp. tularensis Schu S4. Both a WT and MDR strain, which was created to model a hygromycin- and kanamycin-resistant strain, were used. CFU determination was used to calculate the precise IC50of each AR-12 analog compound,and LC50values were used to determine the therapeutic index. OD600titration assays were performed to determine the direct impact against F. tularensis Schu S4 (MIC50). These results are reported in Table 5.

[0105] Structures of AR-12 analogs evaluated for activity against S. enterica and F. tularensis are depicted in Table 6. Table 6

[0106] Structures of the AR-12 analog compounds in Table 5 are depicted in FIG. 6. Compounds 122 and 241, Cutamesine (also known as SA 4503) and BAY 61-3606 respectively, are known compounds.

[0107] The foregoing description and examples have been set forth merely to illustrate the invention and are not meant to be limiting. Since modifications of the described embodiments incorporating the spirit and the substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations within the scope of the claims and equivalents thereof.

Claims

CLAIMS 1. A compound of Formula (I):wherein: A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6; R1is -H, -F, -Cl, -Br, or -I; R, , , R3 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R4is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl; or a substituted or unsubstituted aryl; R5is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6alkyl, or a substituted or unsubstituted C3-C10 cycloalkyl; or a substituted or unsubstituted aryl; R6 is -H, -F, -Cl, -Br, -I, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl;R7is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl,, or; R8 is -H, -F, -Cl, -Br, -I, -a substituted or unsubstituted C1-C6 alkyl, -OR12, or -NR13N14; R9 is -H, -F, -Cl, -Br, -I, -a substituted or unsubstituted C1-C6 alkyl, -OR12, or -NR13N14; R10 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or phenyl; R11is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; R12 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or phenyl; R13 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein: A is N or CR3;B is N or CR4; D is N or CR5; E is N or CR6; R1is -H, -F, -Cl, -Br, or -I;R3 is -H, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCl3, -CHCl2, -CH2Cl, -CBr3, -CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R4 is -H, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCl3, -CHCl2, -CH2Cl, -CBr3, -CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R5is -H, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCl3, -CHCl2, -CH2Cl, -CBr3, -CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, a substituted or unsubstituted C3-C10cycloalkyl, or a substituted or unsubstituted aryl; R6is -H, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCl3, -CHCl2, -CH2Cl, -CBr3, -CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted aryl; R7 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl,, or; R8 is -H, -F, -Cl, -Br, -I, -CF3, -CCl3, -CBr3, -CI3, methyl, ethyl, n-propyl, isopropyl, n- butyl, sec-butyl, tert-butyl, -OR12, or -NR13N14; R9 is -H, -F, -Cl, -Br, -I, -CF3, -CCl3, -CBr3, -CI3, methyl, ethyl, n-propyl, isopropyl, n- butyl, sec-butyl, tert-butyl, -OR12, or -NR13N14;R10is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or phenyl; R11 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; R12 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or phenyl; R13is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14 is -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1, wherein: A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6; R1 is -H, -F, -Cl, -Br, or -I;R3 is -H, -F, -Cl, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted naphthyl, a substituted or unsubstituted anthryl, a substituted or unsubstituted phenanthryl, or a substituted or unsubstituted pyrenyl; R4 is -H, -F, -Cl, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted naphthyl, a substituted or unsubstituted anthryl, a substituted or unsubstituted phenanthryl, or a substituted or unsubstituted pyrenyl; R5is -H, -F, -Cl, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted naphthyl, a substituted or unsubstituted anthryl, a substituted or unsubstituted phenanthryl, or a substituted or unsubstituted pyrenyl; R6is -H, -F, -Cl, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted naphthyl, a substituted or unsubstituted anthryl, a substituted or unsubstituted phenanthryl, or a substituted or unsubstituted pyrenyl; R7 is -H, methyl, ethyl,, R8is -H, -F, -Cl, -CF3, methyl, ethyl, -OR12, or -NR13N14; R9 is -H, -F, -Cl, -CF3, methyl, ethyl, -OR12, or -NR13N14; R10 is -H, methyl, ethyl, or phenyl; R11is -H, methyl, ethyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p- toluenesulfonyl (tosyl, Ts) group; R12is -H, methyl, ethyl, or phenyl; R13is -H, methyl, ethyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9- fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p- toluenesulfonyl (tosyl, Ts) group; and R14 is -H, methyl, ethyl, phenyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert-butyloxycarbonyl (BOC) group, a carbobenzyloxy (Cbz) group, a 9- fluorenylmethyloxycarbonyl (Fmoc) group, a methanesulfonyl (mesyl, Ms) group, and a p- toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

4. The compound of claim 1, wherein: A is N or CR3; B is N or CR4; D is N or CR5; E is N or CR6; R1 is -H or -F;, , , R3is -H, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl;R4is -H, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl; R5is --H, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl; R6 is -H, -CF3, -CHF2, -CH2F, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a substituted or unsubstituted phenyl;R8 is -H or -NR13N14; R9is -H or -NR13N14; R10 is -H, methyl, or phenyl; R11 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert- butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; R13 is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert- butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; and R14is -H, methyl, or a protecting group selected from the group consisting of a benzyl (Bn) group, a formyl group, an acetyl (Ac) group, a benzoyl (Bz) group, a tert- butyloxycarbonyl (BOC) group, a methanesulfonyl (mesyl, Ms) group, and a p-toluenesulfonyl (tosyl, Ts) group; or a pharmaceutically acceptable salt thereof.

5. A compound of claim 1 selected from the group consisting of:;; or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition comprising a therapeutically effective amount of at least one compound of claim 1 and at least one pharmaceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, further comprising at least one other pharmaceutically active agent selected from the group consisting of antibacterial compounds,antiviral compounds, antifungal compounds, antiparasitic compounds, and combinations thereof.

8. The pharmaceutical composition of claim 7, wherein the at least one other pharmaceutically active agent is selected from the group consisting of beta-lactams, floroquinolones, macrolides, aminoglycosides, tetracyclines, and mixtures thereof.

9. The pharmaceutical composition of claim 7, wherein the at least one other pharmaceutically active agent is selected from the group consisting of gentamicin, streptomycin, rapamycin, kanamycin, hygromycin, clindamycin, penicillin, ampicillin, chloroquine, chloramphenicol, linezolid, ciprofloxacin, imipenem, cilastatin, doxycycline, and mixtures thereof.

10. A method of treating an infection caused by a microbial species, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of claim 1.

11. The method of claim 10, wherein the compound is selected from the group consisting of:; or a pharmaceutically acceptable salt thereof.

12. The method of claim 10, further comprising administering to a subject in need thereof a therapeutically effective amount of at least one other pharmaceutically active agent selected from the group consisting of antibacterial compounds, antiviral compounds, antifungal compounds, antiparasitic compounds, and combinations thereof.

13. The method of claim 12, wherein the compound of claim 1 and the at least one other pharmaceutically active agent are administered simultaneously.

14. The method of claim 13, wherein the compound of claim 1 and the at least one other pharmaceutically active agent are administered in the same dosage form.

15. The method of claim 13, wherein the compound of claim 1 and the at least one other pharmaceutically active agent are administered in separate dosage forms.

16. The method of claim 12, wherein the compound of claim 1 and the at least one other pharmaceutically active agent are administered sequentially.

17. The method of claim 12, wherein the at least one other pharmaceutically active agent is selected from the group consisting of beta-lactams, floroquinolones, macrolides, aminoglycosides, tetracyclines, and mixtures thereof.

18. The method of claim 12, wherein the at least one other pharmaceutically active agent is selected from the group consisting of gentamicin, streptomycin, rapamycin, kanamycin, hygromycin, clindamycin, penicillin, ampicillin, chloroquine, chloramphenicol, linezolid, ciprofloxacin, imipenem, cilastatin, doxycycline, and mixtures thereof.

19. The method of claim 10, wherein the infection is caused by Salmonella enterica or Francisella tularensis.

20. The method as in claim 10, wherein the subject is a mammal selected from the group consisting of humans, primates, horses, sheep, pigs, cows, mice, rats, rabbits, dogs, and cats.

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